CRISPR/Cas9-mediated disruption of lipocalins, Ly6g5b, and Ly6g5c causes male subfertility in mice

Abstract

Background: Spermatozoa become mature and competent for fertilization during transit from the caput epididymis to the cauda epididymis. However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis-enriched genes: lipocalin family genes (Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10) and Ly6 family genes (Ly6g5b and Ly6g5c). These genes are evolutionarily conserved in mammals and form clusters on chromosomes 2 and 17 in the mouse, respectively.

Objective: To clarify whether these genes are required for epididymal sperm maturation and acquisition of fertilizing ability, we generated knockout (KO) mice using the CRISPR/Cas9 system and analyzed their phenotype.

Materials and methods: We generated four lines of KO mice: Lcn9 single KO, the lipocalin family quadruple KO (Lcn5, Lcn6, Lcn8, and Lcn10), quintuple KO (Lcn5, Lcn6, Lcn8, Lcn10, and Lcn9), and double KO of Ly6 family genes (Ly6g5b and Ly6g5c).

Results: Although the Lcn9 single KO did not affect male fertility, the quadruple KO and quintuple KO male mice were subfertile and mostly infertile, respectively, with a reduced amount of ADAM3, an essential protein for sperm binding to the zona pellucida. Further analysis revealed that the quintuple KO spermatozoa lack the CMTM2A/B that are required for ADAM3 maturation. Intriguingly, Ly6g5b and Ly6g5c double KO male mice also showed subfertility with reduced sperm ADAM3.

Conclusion: These results suggest epididymal secretory proteins are involved in ADAM3 maturation and acquisition of sperm fertilizing ability.

Keywords: Adam3; epididymis; knockout; sperm maturation; zona pellucida.

Fig. 1.

Multiple gene expression by RT-PCR and phenotype analysis of Lcn9 KO mice. (A) Gene expression of Lcn5, Lcn6, Lcn8, Lcn9, Lcn10, Ly6g5b, and Ly6g5c. Actin β (Actb) was used as the control. Epididymis (Epi). (B) Male fertility. There was no difference in the average pups/plug ratio of WT and KO males (P = 0.68). (C) Cauda epididymal spermatozoa from WT B6D2F1 and Lcn9^del/del mice. Scale bars are 50 μm. (D) Histological analysis of the epididymis with H & E staining. The right panels are enlargements of the dashed areas in the left panels. Scale bars on the left and right panels are 1 mm and 100 μm, respectively.

Fig. 2.

Phenotype analysis of Lcn8-Lcn10 KO mice. (A) Genomic structure of Lcn8, 5, 6, 10, and 9. Gene information was obtained from Ensemble (http://ensembl.org/index.html). (B) Number of pups per plug of WT female mice mated with KO male mice. WT B6D2F1 male mice were used as the control. * P < 0.01. (C) Cauda epididymal spermatozoa from (Lcn8-Lcn10)^wt/del and (Lcn8-Lcn10)^del/del mice. Scale bars are 50 μm. (D) Histological analysis of epididymis with H & E staining. The right panels are enlargements of the dashed areas in the left panels. Scale bars on the left and right panels are 1 mm and 100 μm, respectively. (E) In vitro fertilization ability analysis. (F and G) Sperm-zona pellucida binding assay. The average number of zona pellucida-binding spermatozoa obtained from heterozygote and homozygote mutant mice. Scale bars are 25 μm. * P < 0.01.

Fig. 3.

Phenotype analysis of Lcn8-Lcn10 and Lcn9 KO mice. (A) Value of pups per plug of WT female mice mated with KO male mice. WT B6D2F1 male mice were used as the control. * P < 0.01. (B) Cauda epididymal spermatozoa from (Lcn8-10/9)^wt/del and (Lcn8-10/9)^del/del mice. Scale bars are 50 μm. (C) Histological analysis of the epididymis with H & E staining. The right panels are enlargements of the dashed areas in the left panels. Scale bars on the left and right panels are 1 mm and 100 μm, respectively. (D) In vitro fertilization ability analysis. (E) Sperm-zona pellucida binding assay. Average number of zona pellucida-binding spermatozoa obtained from heterozygote and homozygote mutant mice. * P < 0.01.

Fig. 4.

Phenotype analysis of Ly6g5b/c KO mice. (A) Genomic structure of Ly6g5c and b. Gene information was obtained from Ensemble. (B) Value of pups per plug of WT female mice mated with KO male mice. WT B6D2F1 male mice were used as the control. * P < 0.01. (C) Cauda epididymal spermatozoa from (Ly6g5b/c)^wt/del and (Ly6g5b/c)^del/del mice. Scale bars are 50 μm. (D) Histological analysis of the epididymis with H & E staining. The right panels are enlargements of the dashed areas in the left panels. Scale bars on the left and right panels are 1 mm and 100 μm, respectively. (E) In vitro fertilization ability analysis. (F) Sperm-zona pellucida binding assay. The average number of zona pellucida-binding spermatozoa obtained from heterozygote and homozygote mutant mice. * P < 0.01.

Fig. 5.

Immunoblot analysis. (A-C) Immunoblot analysis for ADAM3. The study was performed using testicular germ cells (TGC) and cauda epididymal spermatozoa obtained from (A) Lcn8-Lcn10, (B) Lcn8-10/9, and (C) Ly6g5b/c mutant mice. BASIGIN was used as the control. (D) Immunoblot analysis for OVCH2 and RNASE10 using protein lysates collected from WT and Lcn8-10/9 KO testis, caput, corpus, and cauda sperm. GAPDH was used as the control. (E) Immunoblot analysis for ADAM1B, ADAM2, CMTM2A, CMTM2B, and IZUMO1 using cauda sperm lysates collected from WT and Lcn8-10/9 KO mice. BASIGIN was used as the control. (F) Schematic view of this study.